BMBMethod:ChemicalTransformation2

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Chemical Transformation of E. coli (heat-shock transformation) Notes by Mike Fisher

  • Thaw frozen competent cells (50ul) on ice (~5 min)
  • Add DNA, then flick to mix
    • Usually 2-5ul for ligations, 1ul (less than 10ng) for completed constructs. Do not exceed 100ng of DNA or 10% of the transformation volume.
    • Be careful not to warm cells
  • Incubate on ice for 30 minutes.
    • This is a minimum, can go longer
  • Heat shock for 45 seconds at 42°C. The temperature doesn't have to be exactly 42°C, but the timing does matter.
  • Immediately put cells back on ice for 2 minutes.
  • Recovery - add 9x volume of SOB or SOC and incubate shaking for 1 hour at 37°C.
    • The recovery step is critical. Recovering for less than 1 hour begins to drastically reduce transformation efficiency
    • If you are transforming bacteria that isn't very competent, recovering for an additional 1-2 hours helps a lot. However, this isn't advisable for ligations.
  • Plate some or all of the cells on LB Agar plates with appropriate antibiotic
  • If plating all of the cells
    • Briefly spin down the cells
      • 3000 x g for 5 minutes
    • Remove all but 50-200 ul of media.
    • Gently resuspend the cell pellet by pipetting
    • Plate on media containing the appropriate antibiotic. A typical LB Agar petri dish lets you plate 50-200μL of liquid. Too little, and spreading is hard. Too much, and you make a mess.